Introduction: Assisted reproductive technology (ART) is associated with an increased risk of monozygotic twinning. This narrative review attempts to summarise the known literature regarding the aetiology, incidence, risk factors, diagnosis, and prognosis of monozygotic twinning following ART. Aetiology: Monozygotic twinning is caused by the splitting of the early embryo during the peri-implantation phase. According to the classical hypothesis, the timing of the split determines the chorionicity and amnionicity, however this has been questioned in recent literature. Incidence and risk factors: The incidence of monozygotic twinning in natural conception appears to be independent of extrinsic factors such as ethnicity and age. The incidence of monozygotic twinning is increased from 0.4% of natural conceptions to around 0.9–2.24% of pregnancies following ART. The available literature supports a role of ovarian stimulation and extended culture to the blastocyst stage in increasing the risk of monozygotic twinning. The impact of maternal age and micromanipulation techniques such as assisted hatching and intra-cytoplasmic sperm injection appear to depend on the stage of the embryo being transferred leading to significant heterogeneity between studies. Diagnosis: The gold standard for diagnosing monozygotic twinning is genetic testing but its cost precludes it from routine widespread use. Most epidemiological studies utilise statistical estimates such as Weinberg’s differential rule and tailored questionnaires. Most studies from ART units have utilised transvaginal sonography for counting the number of gestational sacs or assessing the chorionicity. Prognosis: The prognosis of twins appears to be dependent on the chorionicity and amnionicity and is largely independent of the zygosity.

We report a novel method to profile intrcellular oxygen concentration (icO2) during in vitro mammalian oocyte and preimplantation embryo development using a commercially available multimodal phosphorescent nanosensor (MM2). Abattoir-derived bovine oocytes and embryos were incubated with MM2 in vitro. A series of inhibitors were applied during live-cell multiphoton imaging to record changes in icO2 associated with mitochondrial processes. The uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) uncouples mitochondrial oxygen consumption to its maximum, while antimycin inhibits complex III to ablate mitochondrial oxygen consumption. Increasing oxygen consumption was expected to reduce icO2 and decreasing oxygen consumption to increase icO2. Use of these inhibitors quantifies how much oxygen is consumed at basal in comparison to the upper and lower limits of mitochondrial function. icO2 measurements were compared to mitochondrial DNA copy number analysed by qPCR. Antimycin treatment increased icO2 for all stages tested, suggesting significant mitochondrial oxygen consumption at basal. icO2 of oocytes and preimplantation embryos were unaffected by FCCP treatment. Inner cell mass icO2 was lower than trophectoderm, perhaps reflecting limitations of diffusion. Mitochondrial DNA copy numbers were similar between stages in the range 0.9–4 × 106 copies and did not correlate with icO2. These results validate the MM2 probe as a sensitive, non-toxic probe of intracellular oxygen concentration in mammalian oocytes and preimplantation embryos.